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(Circulation Research. 1995;76:634-644.)
© 1995 American Heart Association, Inc.

Articles

Acetylcholine Elicits a Rebound Stimulation of Ca²⁺ Current Mediated by Pertussis Toxin–Sensitive G Protein and cAMP-Dependent Protein Kinase A in Atrial Myocytes

Yong Gao Wang, Stephen L. Lipsius

From the Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois.

Correspondence to Stephen L. Lipsius, PhD, Department of Physiology, Loyola University Medical Center, 2160 S First Ave, Maywood, IL 60153.

Abstract Cholinergic inhibition of atrial contraction is typically followed by a rebound positive inotropic response. In the present study, we used a nystatin–perforated patch whole-cell recording method to determine whether acetylcholine (ACh) elicits a rebound stimulation of L-type Ca²⁺ current (I_Ca,L) in cat atrial myocytes. ACh (1 µmol/L) decreased basal I_Ca,L (-19±2%). Within 30 s of returning to ACh-free solution, basal I_Ca,L exhibited a rebound increase above the control level (+61±7%) that returned to the control level within 4 to 5 minutes. ACh elicited concomitant changes in cell shortening, ie, a decrease followed by a rebound increase. The EC₅₀ and maximal response of ACh-induced inhibition and rebound stimulation of I_Ca,L were 1.9x10^-9 mol/L and -30%, respectively, and 2.9x10^-8 mol/L and +64%, respectively. All effects of ACh on I_Ca,L were blocked by prior exposure to 1 µmol/L atropine or 100 µmol/L AFDX116 and unaffected by 0.2 µmol/L pirenzepine or 1 µmol/L propranolol. In the presence of ACh, exposure to atropine elicited stimulation of I_Ca,L. ACh-induced inhibition and rebound stimulation of current were independent of external Ca²⁺. Rebound stimulation of I_Ca,L was associated with a negative shift in the voltage dependence of I_Ca,L activation. Inhibition of protein kinase A by 50 µmol/L Rp-cAMPs decreased basal I_Ca,L by 36±1% and abolished the rebound stimulation of I_Ca,L. Forskolin (0.01 µmol/L) or isoproterenol (0.01 µmol/L) had no effect on basal I_Ca,L, but each accentuated the rebound increase in I_Ca,L. When adenylate cyclase was maximally stimulated with 1 µmol/L isoproterenol plus 2 µmol/L forskolin, ACh decreased I_Ca,L but failed to elicit rebound stimulation of I_Ca,L. Milrinone (10 µmol/L) increased basal I_Ca,L by 70±7% and significantly attenuated the rebound stimulation of I_Ca,L. Exposure to 1 mmol/L 8-bromo-cGMP elicited a small decrease in basal I_Ca,L, attenuated ACh-induced inhibition, and enhanced the rebound stimulation of I_Ca,L. Incubation in pertussis toxin prevented all ACh-induced changes in I_Ca,L. Inhibition of nitric oxide synthase by 100 µmol/L N^G-monomethyl-L-arginine (L-NMMA) decreased basal I_Ca,L by -20±5%, prevented ACh-induced inhibition, and markedly attenuated the rebound stimulation of I_Ca,L. We conclude that in cat atrial myocytes ACh acts via M₂ muscarinic receptors and pertussis toxin–sensitive G protein to inhibit basal I_Ca,L and that on withdrawal ACh elicits a rebound stimulation of I_Ca,L. Rebound stimulation of I_Ca,L is mediated via cAMP-dependent protein kinase A enhanced by ACh-induced inhibition of phosphodiesterase. This mechanism contributes directly to the positive inotropic response that follows cholinergic inhibition of atrial contraction.

Key Words: perforated patch • whole-cell patch • isoproterenol • pertussis toxin • phosphodiesterase

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